The present invention relates to assays, screening methods, peptides, mimetics, and methods of use based on the discovery and characterisation of an interaction between Ku70 and Ku80 and DNA-PKcs. More particularly, aspects of the invention are based around peptide fragments of Ku70 and Ku80. The invention relates to numerous cellular processes which are of interest in therapeutic contexts.
Ku is a protein that is found in a wide range of organisms, ranging from Saccharomyces cerevisiae to man (Dynan and Yoo, 1998). It is expressed in all human tissues examined. Ku comprises two tightly-associated subunits of about 69 kDa and about 83 kDa. These are termed Ku70 and Ku80 (or Ku86), respectively. Although some information has been obtained regarding the regions of the Ku polypeptides that interact with one another (Cary et al., 1998; Jin and Weaver, 1997; Koike et al., 1998; Osipovich et al., 1997; Wang et al., 1998a; Wang et al., 1998b; Wu and Lieber, 1996), little is known about the precise sites of interaction and the molecular mechanism underlying it.
The most highly characterised function of Ku at the biochemical level is its ability to bind avidly to certain disruptions of the DNA double helix in a sequence independent fashion. The most well studied example of such a disruption is the DNA double-strand break (DSB; Blier et al., 1993; Devries et al., 1989; Mimori and Hardin, 1986). Other discontinuities that are recognised by Ku include single-strand breaks in the sugar-phosphate backbone of double-stranded DNA (dsDNA), and DNA single-strand to double-strand transitions, such as those that occur in hairpin loops or single-stranded gaps in a dsDNA molecule (Blier et al., 1993; Falzon et al., 1993). Once bound to a dsDNA end, Ku can move to internal positions in the DNA in an ATP-independent fashion (Paillard and Strauss, 1991; Devries et al., 1989). Ku has also been reported to be capable of sequence-specific DNA interactions (Giffin et al., 1996): for a review see (Dynan and Yoo, 1998). It has also been reported that Ku70 and possibly Ku80 are capable of interacting with DNA in the absence of their heterodimerisation partner (Chou et al., 1992; Wang et al., 1994). It has also been demonstrated that Ku heterodimers bound to DNA are able to specifically associate with one another (Cary et al., 1997).
When complexed with DNA, Ku can interact with an approximately 460 kDa polypeptide, the DNA-dependent protein kinase catalytic subunit (DNA-PKcs). DNA-PKcs is a member of the PI 3-kinase-like (PIKL) protein kinase family (Hartley et al., 1995) and, together with Ku and DNA, forms a catalytically active DNA-PK complex with Ser/Thr kinase activity (Dvir et al., 1992; Gottlieb and Jackson, 1993; Suwa et al., 1994).
Little is currently known about how Ku interacts with DNA-PKcs, although the fact that it is apparently unable to bind DNA-PKcs in the absence of DNA (Suwa et al., 1994) suggests that DNA binding by Ku induces a conformational change that permits the DNA-PKcs interaction. In particular, previous studies have provided little information about the regions of the Ku heterodimer which are involved in the DNA-PKcs interaction.
It has been shown that, under certain conditions, DNA-PKcs can bind to dsDNA ends and become activated in the absence of Ku (Hammarsten and Chu, 1998; Yaneva et al., 1997). Thus, whilst allosteric activation of DNA-PKcs by Ku might occur, it appears that direct interactions between DNA-PKcs and DNA can be sufficient to activate the kinase in vitro. This suggests that contacts between DNA-PKcs and DNA play an important role in DNA-PK activation, even in the presence of Ku.
A breakthrough in the understanding of DNA-PKcs/Ku function came with the discovery that defects in these proteins are associated with a subset of mutant mammalian cell lines that are defective in DNA DSB rejoining, and are profoundly sensitive to ionising radiation and other agents that generate DNA DSBs as their principal lethal lesion (Jackson, S. P., et al (1995) TIBS 20, 412-415; Critchlow, S. E., et al (1998) TIBS 23, 394-398). Indeed, the mutant phenotypes of these cells are corrected by the introduction of the appropriate Ku or DNA-PKcs expression vector, and recent work using extracts of mammalian or Xenopus laevis cells has provided evidence for a direct involvement of Ku and DNA-PKcs in DNA DSB rejoining (Baumann and West, 1998; Labhart, P., (1999) Mol. Cell. Biol. 19, 2585-2593). Furthermore, DNA-PK catalytic activity has been implicated at an early stage of DNA DSB repair in Xenopus cell-free extracts (Gu et al., 1996; Gu et al., 1998) and for radiation-induced DNA repair in cultured human cells (Okayasu, R., et al (1998) Radiat. Res. 149, 440-445). Coupled with the fact that Ku displays a very high affinity for dsDNA ends in vitro, these data suggest that DNA-PK functions directly in the recognition and resolution of radiation-induced DNA DSBs in vivo.
Cells deficient in DNA-PKcs, Ku80, or Ku70 are also severely impaired in V(D)J recombination, a site-specific genomic rearrangement process that takes place in the developing vertebrate immune system to help generate the vast antigen recognition capacity of antibody and T-cell receptor molecules (Jackson, S. P., et al (1995) TIBS 20, 412-415; Critchlow, S. E., et al (1998) TIBS 23, 394-398). This process requires the production of DNA DSBs between the recombining gene segments by the RAG1/RAG2 proteins (Jackson, S. P., et al (1995) TIBS 20, 412-415; Critchlow, S. E., et al (1998) TIBS 23, 394-398) and the subsequent rejoining of the DNA ends via DNA-PK-dependent mechanisms. For a single DNA rearrangement between two coding segments (V, D, or J regions) to occur, a join between the two coding sequences (known as the coding join) and one between the two non-coding signal ends (the signal join) are made. Interestingly, Ku is essential for both types of join, whereas DNA-PKcs appears to be required only for coding joins and plays a non-essential and variable role in the generation of signal joins (Bogue et al., 1998). This suggests that, at least for the repair of a sub-set of DNA DSBs, Ku is able to function in the absence of DNA-PKcs.
Consistent with the Ku-associated DNA DSB repair pathway being highly conserved throughout eukaryotic evolution, Ku is found in S. cerevisiae and is essential for repair of DNA DSBs by the pathway of non homologous end-joining (Boulton and Jackson, 1996b; Siede et al., 1996 and see Critchlow and Jackson, 1998 for review). Perhaps surprisingly there is no clear orthologue of DNA-PKcs encoded by the fully-sequenced S. cerevisiae genome. Thus, in yeast, Ku carries out DNA-repair functions independently of DNA-PK. Although it is possible that the functions of mammalian DNA-PKcs are assumed by other members of the PIKL protein kinase familly, such as Mec1p and/or Tel1p, there is no evidence to suggest that these interact physically or genetically with Ku.
Interestingly, S. cerevisiae Ku has also been shown to play important roles in telomere length maintenance, and in the transcriptional silencing of genes placed close to telomeric DNA (Boulton and Jackson, 1996a; Boulton and Jackson, 1998 Porter et al., 1996).
The present inventors have investigated interactions between Ku70 and Ku80, and between the two Ku subunits and DNA-PKcs. The data presented herein lead to the conclusion that the two Ku subunits are structurally and functionally related to each other, and appear to associate via a pseudo-homodimerisation mechanism. Furthermore, the work demonstrates that the extreme C-terminus of Ku80 plays an important role in the interaction between Ku and DNA-PKcs. These results provide for modulation of the structure and physiological functions of DNA-PKcs and Ku, for instance by means of peptides corresponding to conserved regions in Ku70 or Ku80 and/or regions of interaction between Ku70 and Ku80 and/or other molecules such as DNA-PKcs, and allow for postulation of a model for the evolution of the DNA-PK complex.
Based on the experimental work and discussion herein the invention is further concerned with assays and methods for identifying homologues and orthologues of Ku polypeptides.
The present inventors have analysed the amino acid sequences of Ku70 and Ku80 polypeptides from a diverse range of species, and have identified six regions of sequence homology between them. These regions are referred to as Homology regions 1-6 (HRs 1-6) and are shown in FIG. 1 as shaded boxes. Corresponding regions from other sequences, e.g. from mammalian, e.g. rodent, e.g. mouse, hamster, sequences are easily identifiable by those skilled in the art using the work, discussion and Figures presented herein. References to, and accession numbers of certain Ku70 and Ku80 sequences are provided in Table 1. Corresponding regions from the mouse sequences are provided in Table 2.
In one aspect the invention provides a peptide or polypeptide consisting of any of the amino acid sequences of any of the Homology Regions shown in FIG. 1, or a peptide or polypeptide consisting of a variant of such a sequence. Variants are discussed elsewhere herein. Variants include corresponding sequences from other animals, e.g. from mammals, e.g. from rodents. Examples of variants are the amino acid sequences of the Homology regions of mouse and hamster Ku70 or Ku80 sequences. The homology regions of the mouse sequences are provided in Table 2.
Variants of the sequences of the Homolgy Regions of human Ku70 and human Ku80 include the following: for Ku70 HR3, sequences which lack one or more residues corresponding to L420, D421, D422; for Ku80 HR3, sequences which lack one or more residues corresponding to N415, Y416, E417; for Ku70 HR5, sequences which lack one or more residues corresponding to T449, E450, K451, I452; for Ku80 HR5 sequences which lack one or more residues corresponding to S441, K442; for Ku70 HR6, sequences which lack one or more residues corresponding to E501, Q502; for Ku80 HR6 sequences which comprise one or more residues corresponding to L505, P506.
A peptide or polypeptide consisting of any of the amino acid sequences of any of the Homology Regions shown in FIG. 1 or a peptide or polypeptide consisting of a variant of such a sequence, (a xe2x80x9cKu Homology Region Peptidexe2x80x9d), may be used to establish antibodies which in turn may be used in the identification of Ku polypeptides, or in the determination of the presence and/or quantity of Ku polypeptide in a test sample. Such antibodies form a further aspect of the invention in their own right. Methods of generating such antibodies, e.g. by raising them in a host organism or selection from an immunoglobulin expression libraray, are discussed elsewhere herein, and are well known to those having skill in the art.
By way of example, antibodies which bind to peptides or polypeptides consisting of any of the amino acid sequences of Ku Homology Region Peptide may be used to probe samples or expression libraries for Ku polypeptides, e.g. for the Ku polypeptide used in their generation, or for a cross-reactive Ku polypeptide. Binding of antibodies to Ku polypeptide may be determined by any method known to those skilled in the art. Methods for determining binding and interaction are discussed herein. Samples may be tissue or cellular (e.g. nuclear) extracts, or in vitro translation systems expressing nucleic acids of interest. Expression libraries may be bacterial or phage libraries.
Methods of determining the presence of, and optionally quantifying the amount of a Ku polypeptide in a test sample may have a diagnostic purpose, e.g. in the diagnosis of any medical condition discussed herein (e.g. in a disease or disorder associated with loss of a Ku polypeptide). Such methods may also be used to evaluate a therapy to treat such a condition.
Ku Homology Region Peptides as defined herein correspond to regions of Ku that are important for Ku function, e.g. for binding to proteins or nucleic acid, or for maintaining tertiary structure. Accordingly, they may interact with other molecules and may be used to disrupt interaction between Ku and such other molecules. For instance a peptide corresponding to HR5 or HR6 may be used to interfere with interaction between Ku70 and Ku80, with functional or biological consequences as discussed. Similarly, a peptide corresponding to any of HR1, HR2, HR3 or HR4 may be used to modulate a Ku-dependent activity or function which may have biological and/or therapeutic consequences.
In various further aspects, the present invention provides for nucleic acid encoding a polypeptide consisting of a Ku Homology Region Peptide, for vectors containing such nucleic acid, and for host cells containing such nucleic acid or such vectors. Nucleic acids, vectors and host cells are discussed further elsewhere herein, and are useful in production of peptides and polypeptides by recombinant means.
Further peptides of interest in the present invention correspond to the region of Ku80 identified herein as being necessary and sufficient for binding to DNA-PKCS, and may include or consist of the amino acid sequence EGGDVDDLLDMI (SEQ ID NO:1). Aspects of the invention based on this include assay methods for determining binding between Ku80 or EGGDVDDLLDMI (SEQ ID NO:1) on the one hand and DNA-PKCS on the other, identification of binding partners, and assay methods for agents that modulate, especially disrupt, such binding and which have therapeutic potential.
As used herein, the term xe2x80x9cEGGDVDDLLDMI peptidexe2x80x9d refers to a peptide which comprises or consists of the amino acid sequence EGGDVDDLLDMI (SEQ ID NO:1) and which has the ability to bind DNA-PKCS. The term also refers to which comprise a variant of the sequence amino acid sequence EGGDVDDLLDMI (SEQ ID NO:1) and which have the ability to bind DNA-PKCS. Variants are discussed elsewhere herein. Variants of the amino acid sequence EGGDVDDLLDMI (SEQ ID NO:1) include the corresponding regions of the related sequences shown in FIG. 6 (SEQ ID NOS:16-18) (multiple sequence alignment generated using the program xe2x80x9cpileupxe2x80x9d from the sequence analysis xe2x80x9cWisconsin Package, version 8.1xe2x80x9d (Program Manual for the Wisconsin Package, Version 8, September 1994, Genetics Computer Group: Gap creation 3.00; Gap extension 0.10).
EGGDVDDLLDMI peptides may for example be: (i) the amino acid sequence EGGDVDDLLDMI (SEQ ID NO:1) or a variant thereof; or (ii) the peptide of (i) fused to a heterologous amino acid sequence (see below), i.e. a sequence to which it is not naturally fused in Ku80, or (iii) the peptide of (i) fused to a sequence with which it is naturally fused in wild-type Ku80, but in a peptide or polypeptide which has the ability to bind to DNA-PKCS.
A peptide in accordance with any aspect of the present invention may include one or more heterologous amino acids joined to the specified peptide. By xe2x80x9cheterologousxe2x80x9d is meant not occurring in a Ku80 or Ku70 polypeptide joined by a peptide bond without intervening amino acids to the relevant specified peptide, that is to say usually a chain of amino acids which is not found naturally joined to the specified peptide at the position of fusion in the peptide of the invention. Usually where heterologous amino acids are included, the contiguous sequence of amino acids does not occur within Ku70 and/or Ku80, and may include or be 5 or more, preferably 10 or more, more preferably 15 or more, 20 or more or 30 or more amino acids with a sequence which does not occur contiguously in Ku70 and/or Ku80
A peptide or polypeptide according to the invention may be about 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23-25, 25-30, 30-35, 35-40, 40-45, 45-50, 50-60, 60-70, 70-80, 80-90, 90-100, 100-125, 125-150, 150-175, 175-200, 200-250, 250-300, 350-400, 400-450 amino acids in length. It may be about 22 amino acids or less, 69 amino acids or less, 137 amino acids or less, 208 amino acids or less, 259 amino acids or less, 314 amino acids or less, 394 amino acids or less in length. A EGGDVDDLLDMI peptide of the invention is able to bind DNA-PKcs and will be able to bind xe2x80x9cDNA-PKcs-like polypeptidesxe2x80x9d, meaning not only the human DNA-dependent protein kinase catalytic subunit DNA-PKcs and its homologues and orthologues in other species, for example in mouse, horse, or Xenopus laevis, but also variants of those DNA-PKcs polypeptides, and polypeptides comprising an amino acid sequence which shares at least 30% amino acid sequence homology with the amino acid sequence of human DNA-PKcs or a homologue or orthologue thereof, more preferably at least 35% sequence homology, more preferably at least 40% sequence homology, more preferably at least 50% sequence homology, more preferably at least 70% sequence homology, more preferably at least 80% sequence homology, still more preferably at least 90% sequence homology with the amino acid sequence of human DNA-PKcs or a homologue or orthologue thereof. The amino acid sequence of human DNA-PKcs is disclosed in Dynan and Yoo, 1998 and has the database accession number U47077. Sequence homology is defined elsewhere herein. The term DNA-PKcs-like polypeptide may be used therefore to refer to related enzymes, e.g. other kinases of the PIKL family, e.g. ATM, ATR, FRAP. The term xe2x80x9cDNA-PKCS-like polypeptidexe2x80x9d also encompasses alleles, mutants, derivatives and fragments of human DNA-PKcs or a homologue or orthologue thereof.
Aspects of the present invention provide for the use of a peptide, whether a Ku Homology Region Peptide or an EGGDVDDLLDMI peptide or polypeptide, in screening or searching for and/or obtaining/identifying a binding partner, such as for an EGGDVDDLLDMI peptide a DNA-PKcs-like polypeptide which has the ability to bind to said EGGDVDDLLDMI.
Thus, a further aspect of the invention provides a screening or assay method for identifying an agent which binds to a peptide of the invention, or for determining binding of an peptide of the invention to an agent of interest may include:
(a) bringing a test substance into contact with said peptide; and
(b) determining binding of the test substance to said peptide.
In one embodiment, a screening or assay method for identifying an agent which binds to an EGGDVDDLLDMI peptide, or for determining binding of an EGGDVDDLLDMI peptide to an agent of interest may include:
(a) bringing a test substance into contact with said EGGDVDDLLDMI peptide; and
(b) determining binding of the test substance to said EGGDVDDLLDMI polypeptide.
A test substance which proves to be an agent which binds to an EGGDVDDLLDMI peptide may be, for example, a DNA-PKCS-like polypeptide or an antibody.
In other embodiments the peptide is a Ku Homology Region Peptide, as disclosed.
The invention may be used to identify one or more regions of a polypeptide or other substance involved in an in vivo or an in vitro interaction with a region of Ku70 or Ku80 corresponding to a Homology Region as disclosed or the sequence EGGDVDDLLDMI (SEQ ID NO:1). This may involve identifying sequence motif(s) in a polypeptide which is/are involved in the interaction of polypeptide with the relevant amino acid sequence in Ku80 or Ku70. Various fragments of a DNA-PKCS-like polypeptide may be used in the above assays, e.g. fragments generated by N-terminal and/or C-terminal deletions of the full DNA-PKCS-like polypeptide sequence.
Related aspects of the present invention provide the use of an peptide sequence of the invention for determining the presence in a test sample of a binding partner which has the ability to bind to said sequence, and the use of an agent which binds a peptide of the invention for determining the presence in a test sample of a Ku70 or Ku80 peptide or polypeptide, such as an EGGDVDDLLDMI polypeptide.
A method for determining the presence in a test sample of an agent, e.g. polypeptide such as an antibody, which has the ability to bind to a peptide of the invention, may include:
(a) bringing a peptide of the invention into contact with the test sample; and
(b) determining binding of the peptide to an agent if present in the test sample.
A method for determining the presence in a test sample of a peptide of the invention, may include:
(a) bringing the an agent, e.g. polypeptide such as an antibody, into contact with the test sample; and
(b) determining binding of the agent to a substance in the test sample.
A method for determining the presence in a test sample of any agent or substance may include quantifying the amount of the agent or substance in the sample.
Methods of determining the presence of an agent or substance in a test sample may have a diagnostic purpose, e.g. in the diagnosis of any medical condition discussed herein (e.g. in a disease or disorder associated with reduction or loss of a Ku-dependent function or biological activity). Such methods may also be used to evaluate a therapy to treat such a condition.
Methods of determining binding and/or interaction in any method described herein are discussed below.
A test agent or substance employed in accordance with the present invention may be a natural or synthetic chemical compound.
A test agent or substance may be 0.005-0.01% pure, 0.01-0.05% pure, 0.05%-0.1% pure, 0.1-0.5% pure, 0.5-1% pure, 1-5% pure. pure, 5-10% pure, 10-20% pure, 20-30% pure, 30-40% pure, 40-50% pure, 50-60% pure, 60-70% pure, 70-80% pure, 80-90% pure, 90-95% pure, or substantially pure.
A screening or assay method may include purifying and/or isolating a test substance and/or substance of interest from a mixture or extract, i.e. reducing the content of at least one component of the mixture or extract, e.g. a component with which the test substance or substance of interest is naturally associated. The screening or assay method may include determining the ability of one or more fractions of a test mixture or extract to bind to peptide of the invention. The purifying and/or isolating may employ any method known to those skilled in the art.
The precise format of any of the screening or assay methods of the present invention may be varied by those of skill in the art using routine skill and knowledge. The skilled person is well aware of the need to employ appropriate control experiments.
A peptide of the invention and any agent identified by any one of the methods provided by the present invention may be isolated and/or purified and/or further investigated and/or manufactured. Various methods and uses of such compounds are discussed elsewhere herein.
The inventors have identified a number of EGGDVDDLLDMI polypeptides which can bind to human DNA-PKcs. These sequences form the basis of further aspects of the present invention.
Accordingly, the invention provides the EGGDVDDLLDMI polypeptides Ku80xcex94N1, Ku80xcex94N2, Ku80xcex94N4, Ku80xcex94N5, Ku80xcex94N6, Ku80xcex94N7, Ku80xcex94N8 as shown in FIG. 2 and variants thereof that retain ability to bind to a DNA-PKcs-like polypeptide.
As used herein, variants of a stated amino acid sequence may have an amino acid sequence which shares at least about 30%, or 40%, or 50%, or 60%, or 70%, or 75%, or 80%, or 85%, 90% or 95% homology with the stated sequence. Homology is defined elsewhere herein.
A variant may differ by one or more amino acid residues from the stated sequence, by one or more of addition, insertion, deletion and substitution of one or more amino acid residues. It may include 1, 2, 3, 4, 5, or greater than 5 amino acid alterations such as substitutions with respect to the stated sequence.
A variant of a polypeptide for which the sequence is known or disclosed herein may in certain embodiments be the same length or shorter than that sequence. In other embodiments the polypeptide (e.g. a DNA-PKcs-like polypeptide or an EGGDVDDLLDMI polypeptide or a polypeptide consisting of any of the amino acid sequences of any of the Homology Regions shown in FIG. 1) may be included in a larger polypeptide. For example, 1, 2, 3, 4 or 5, 10, 20 or more additional amino acid residues, adjacent to a native form of the specific polypeptide or heterologous thereto may be included at one end or both ends of the polypeptide.
Variants include mutants, alleles, derivatives and fragments of the stated sequence. Derivatives of polypeptides include the polypeptide linked to a coupling partner, e.g. an effector molecule, a label, a drug, a toxin and/or,a carrier or transport molecule, a nd/or a targeting molecule such as an antibody or binding fragment thereof or other ligand. Techniques for coupling to both peptidyl and non-peptidyl coupling partners are well known in the art. In one embodiment, the carrier molecule is a 16 amino acid peptide sequence derived from the homeodomain of Antennapedia (e.g. as sold under the name xe2x80x9cPenetratinxe2x80x9d), which can be coupled to a peptide via a terminal Cys residue. The xe2x80x9cPenetratinxe2x80x9d molecule and its properties are described in WO 91/18981.
As is well-understood, homology at the amino acid level is generally in terms of amino acid similarity or identity. Similarity allows for xe2x80x9cconservative variationxe2x80x9d, i.e. substitution of one hydrophobic residue such as isoleucine, valine, leucine or methionine for another, or the substitution of one polar residue for another, such as arginine for lysine, glutamic for aspartic acid, or glutamine for asparagine. Homology may be taken over the full-length of a sequence or over a part, such as 20, 30, 40, 50, 60, 70, 80, 90, 100, 120, 150, 200 contiguous nucleotides or amino acids. That two nucleotide sequences are said to share xe2x80x9chomologyxe2x80x9d or be xe2x80x9chomologousxe2x80x9d is based on sequence comparison. Any phylogenetic relationship is irrelevant for this. Those skilled in the art routinely refer to homology between nucleotide sequences with no implication for evolutionary origin. Two homologous nucleotide sequences may also be said to be xe2x80x9csimilarxe2x80x9d or have a certain per centage similarity or a certain per centage identity.
In general it is not critical which of the various standard algorithms are used to determine how homologous two nucleotide sequences are with one another. A preferred algorithm may be GAP, which uses the alignment method of Needleman and Wunsch (J. Mol. Biol. (1970) 48, 443-453) and is included in the Program Manual or the Wisconsin Package, Version 8, September 1994, Genetics Computer Group, 575 Science Drive, Madison, Wis., USA). In the absence of instructions to the contrary, the skilled person would understand to use the default parameters with the aim of maximizing alignment, with a gap creation penalty=12 and gap extension penalty=4.
Similarity or homology (the terms are used interchangeably) or identity may be as defined and determined by the TBLASTN program, of Altschul et al. (1990) J. Mol. Biol. 215: 403-10, or BestFit, which is part of the Wisconsin Package, Version 8, September 1994, (Genetics Computer Group, 575 Science Drive, Madison, Wis., USA, Wisconsin 53711). Preferably sequence comparisons are made using FASTA and FASTP (see Pearson and Lipman, 1988. Methods in Enzymology 183: 63-98). Parameters are preferably set, using the default matrix, as follows: Gapopen (penalty for the first residue in a gap): xe2x88x9212 for proteins/xe2x88x9216 for DNA; Gapext (penalty for additional residues in a gap): xe2x88x922 for proteins/xe2x88x924 for DNA; KTUP word length: 2 for proteins/6 for DNA.
Nucleic acid sequence homology may be determined by means of selective hybridisation between molecules under stringent conditions.
Preliminary experiments may be performed by hybridising under low stringency conditions. For probing, preferred conditions are those which are stringent enough for there to be a simple pattern with a small number of hybridisations identified as positive which can be investigated further.
For example, hybridizations may be performed, according to the method of Sambrook et al. (below) using a hybridization solution comprising: 5xc3x97SSC (wherein xe2x80x98SSCxe2x80x99=0.15 M sodium chloride; 0.15 M sodium citrate; pH 7), 5xc3x97Denhardt""s reagent, 0.5-1.0% SDS, 100 xcexcg/ml denatured, fragmented salmon sperm DNA, 0.05% sodium pyrophosphate and up to 50% formamide. Hybridization is carried out at 37-42xc2x0 C. for at least six hours. Following hybridization, filters are washed as follows: (1) 5 minutes at room temperature in 2xc3x97SSC and 1% SDS; (2) 15 minutes at room temperature in 2xc3x97SSC and 0.1% SDS; (3) 30 minutes-1 hour at 37xc2x0 C. in 1xc3x97SSC and 1% SDS; (4) 2 hours at 42-65xc2x0 C. in 1xc3x97SSC and 1% SDS, changing the solution every 30 minutes.
One common formula for calculating the stringency conditions required to achieve hybridization between nucleic acid molecules of a specified sequence homology is (Sambrook et al., 1989): Tm=81.5xc2x0 C.+16.6Log[Na+]+0.41(% G+C)xe2x88x920.63(% formamide)xe2x88x92600/#bp in duplex.
As an illustration of the above formula, using [Na+]=[0.368] and 50-% formamide, with GC content of 42% and an average probe size of 200 bases, the Tm is 57xc2x0 C. The Tm of a DNA duplex decreases by 1-1.5xc2x0 C. with every 1% decrease in homology. Thus, targets with greater than about 75% sequence identity would be observed using a hybridization temperature of 42xc2x0 C. Such a sequence would be considered substantially homologous to the nucleic acid sequence of the present invention.
It is well known in the art to increase stringency of hybridisation gradually until only a few positive clones remain. Other suitable conditions include, e.g. for detection of sequences that are about 80-90% identical, hybridization overnight at 42xc2x0 C. in 0.25M Na2HPO4, pH 7.2, 6.5% SDS, 10% dextran sulfate and a final wash at 55xc2x0 C. in 0.1xc3x97SSC, 0.1% SDS. For detection of sequences that are greater than about 90% identical, suitable conditions include hybridization overnight at 65xc2x0 C. in 0.25M Na2HPO4, pH 7.2, 6.5% SDS, 10% dextran sulfate and a final wash at 60xc2x0 C. in 0.1xc3x97SSC, 0.1% SDS.
Polypeptides in accordance with and for use in the present invention, e.g. EGGDVDDLLDMI polypeptides or DNA-PKcs-like polypeptides or peptides or polypeptides consisting of a Ku Homology Region peptide as defined, may be generated wholly or partly by chemical synthesis, in accordance with well-established techniques, such as standard liquid or, preferably, solid-phase peptide synthesis methods, general descriptions of which are broadly available (see, for example, in J. M. Stewart and J. D. Young, Solid Phase Peptide Synthesis, 2nd edition, Pierce Chemical Company, Rockford, Ill. (1984), in M. Bodanzsky and A. Bodanzsky, The Practice of Peptide Synthesis, Springer Verlag, New York (1984); and Applied Biosystems 430A Users Manual, ABI Inc., Foster City, Calif.), or they may be prepared in solution, by the liquid phase method or by any combination of solid-phase, liquid phase and solution chemistry.
Another convenient way of producing peptides and polypeptides according to or for use in the present invention is to express encoding nucleic acid, by use of nucleic acid in an expression system.
Nucleic acid molecules encoding a peptide or polypeptide as defined herein in accordance with the present invention represent further aspects of the present invention in their own right.
Generally, nucleic acid according to, or for use in, the present invention is provided as an isolate, in isolated and/or purified form, or free or substantially free of material with which it is naturally associated, such as free or substantially free of nucleic acid flanking the gene in the (e.g. human) genome, except possibly one or more regulatory sequence(s) for expression. Nucleic acid may be wholly or partially synthetic and may include genomic DNA, cDNA or RNA.
Nucleic acid sequences encoding a peptide or polypeptide according to the invention, may be readily prepared by the skilled person using the information and references contained herein and techniques known in the art (for example, see Sambrook, Fritsch and Maniatis, xe2x80x9cMolecular Cloning, A Laboratory Manual, Cold Spring Harbor Laboratory Press, 1989, and Ausubel et al, Short Protocols in Molecular Biology, John Wiley and Sons, 1992). These techniques include (i) the use of the polymerase chain reaction (PCR) to amplify samples of such nucleic acid, e.g. from genomic sources, (ii) chemical synthesis, or (iii) preparing cDNA sequences.
DNA encoding a peptide or polypeptide according to the invention may be generated and used in any suitable way known to those of skill in the art, including taking encoding DNA, identifying suitable restriction enzyme recognition sites either side of the portion to be expressed, and cutting out said portion from the DNA. The portion may then be operably linked to a suitable promoter in a standard commercially available expression system. Another recombinant approach is. to amplify the relevant portion of the DNA with suitable PCR primers.
Modifications to a nucleic acid sequence may be made, e.g. using site directed mutagenesis, to lead to the production of modified forms of a polypeptide, e.g. an EGGDVDDLLDMI polypeptide, e.g. a mutant form of such a sequence, or to take account of codon preference in the host cells used to express the nucleic acid.
In order to obtain expression of the nucleic acid sequences of the invention, the sequences may be incorporated in a vector having one or more control sequences operably linked to the nucleic acid to control its expression. Vectors may contain appropriate regulatory sequences, including promoter sequences, terminator fragments, polyadenylation sequences, enhancer sequences, marker genes and other sequences as appropriate, e.g. nucleic acid sequences so that the polypeptide or peptide is produced as a fusion and/or nucleic acid encoding secretion signals so that the polypeptide produced in the host cell is secreted from the cell. Vectors may be plasmids, viral e.g. phage, or phagemid, as appropriate. For further details see, for example, Molecular Cloning: a Laboratory Manual: 2nd edition, Sambrook et al., 1989, Cold Spring Harbor Laboratory Press. Many known techniques and protocols for manipulation of nucleic acid, for example in preparation of nucleic acid constructs, mutagenesis, sequencing, introduction of DNA into cells and gene expression, and analysis of proteins, are described in detail in Current Protocols in Molecular Biology, Ausubel et al. eds., John Wiley and Sons, 1992.
A peptide or polypeptide according to the invention may then be obtained by transforming the vectors into host cells in which the vector is functional, culturing the host cells so that the encoded peptide or polypeptide, e.g. EGGDVDDLLDMI polypeptide, is produced and recovering the sequence from the host cells or the surrounding medium.
A further aspect of the present invention provides a host cell containing heterologous nucleic acid as disclosed herein.
Systems for cloning and expression of polypeptides in a variety of different host cells are well known. Suitable host cells include bacteria, eukaryotic cells such as mammalian and yeast, and baculovirus systems. Mammalian cell lines available in the art for expression of a heterologous polypeptide include Chinese hamster ovary cells, HeLa cells, baby hamster kidney cells, COS cells and many others. A common, preferred bacterial host is E. coli. 
The nucleic acid of the invention may be integrated into the genome (e.g. chromosome) of the host cell. Integration may be promoted by inclusion of sequences which promote recombination with the genome, in accordance with standard techniques. The nucleic acid may be on an extra-chromosomal vector within the cell, or otherwise identifiably heterologous or foreign to the cell.
Thus, a host cell containing nucleic acid according to the present invention, e.g. as a result of introduction of the nucleic acid into the cell or into an ancestor of the cell (which introduction may take place in vivo or ex vivo), may be comprised (e.g. in the soma) within an organism which is an animal, particularly a mammal, which may be human or non-human, such as rabbit, guinea pig, rat, mouse or other rodent, cat, dog, pig, sheep, goat, cattle or horse, or a bird, such as a chicken.
Genetically modified or transgenic animals or birds comprising such a cell are also provided as further aspects of the present invention.
This may have a therapeutic aim. (Gene therapy is discussed elsewhere herein). Also, the presence of a mutant, allele, derivative or other variant sequence within cells of an organism may allow the organism to be used as a model in studying substances which modulate binding of a peptide to a binding partner, e.g. an EGGDVDDLLDMI polypeptide to a DNA-PKCS-like polypeptide. Conveniently, however, at least preliminary assays for such substances may be carried out in vitro, that is within host cells or in cell-free systems. Where an effect of a test compound is established on cells in vitro, those cells or cells of the same or similar type may be grafted into an appropriate host animal for in vivo testing.
A further aspect provides a method which includes introducing a nucleic acid molecule of the invention into a host cell. The introduction, which may (particularly for in vitro introduction) be generally referred to without limitation as xe2x80x9ctransformationxe2x80x9d, may employ any available technique. For eukaryotic cells, suitable techniques may include calcium phosphate transfection, DEAE-Dextran, electroporation, liposome-mediated transfection and transduction using retrovirus or other virus, e.g. vaccinia or, for insect cells, baculovirus. For bacterial cells, suitable techniques may include calcium chloride transformation, electroporation and transfection using bacteriophage. As an alternative, direct injection of the nucleic acid could be employed.
Marker genes such as antibiotic resistance or sensitivity genes may be used in identifying clones containing nucleic acid of interest, as is well known in the art. The introduction may be followed by causing or allowing expression from the nucleic acid, e.g. by culturing host cells (which may include cells actually transformed although. more likely the cells will be descendants of the transformed cells) under conditions for expression of the gene, so that the encoded polypeptide is produced. If the polypeptide is expressed coupled to an appropriate signal leader peptide it may be secreted from the cell into the culture medium. Following production by expression, a polypeptide may be isolated and/or purified from the host cell and/or culture medium, as the case may be, and subsequently used as desired, e.g. in the formulation of a composition which may include one or more additional components, such as a pharmaceutical composition which includes one or more pharmaceutically acceptable excipients, vehicles or carriers (e.g. see below).
Introduction into target cells of nucleic acid of the present invention may take place in vivo by way of gene therapy, for instance to modulate e.g. disrupt or interfere with, binding of an EGGDVDDLLDMI polypeptide to a DNA-PKcs-like polypeptide. In light of the above, the present invention also provides a method of making a peptide or polypeptide as defined, the method including expression from nucleic acid encoding the peptide or polypepitde. This may conveniently be achieved by growing a host cell containing the nucleic acid in culture under appropriate conditions which cause or allow expression of the peptide or polypeptide. Note however that expression may also be carried out in in vitro systems, e.g. reticulocyte lysate, as discussed elsewhere herein.
As discussed above, the present invention provides EGGDVDDLLDMI polypeptides, methods of identifying further EGGDVDDLLDMI polypeptides which have the ability to bind to a DNA-PKcs-like polypeptide and methods of identifying DNA-PKCS-like polypeptides which have the ability to bind to an EGGDVDDLLDMI polypeptide.
EGGDVDDLLDMI polypeptides having the ability to bind to a DNA-PKcs-like polypeptide will have the ability to modulate, e.g. interfere with, binding of that DNA-PKcs-like poylpeptide to proteins harbouring that EGGDVDDLLDMI polypeptide or variants thereof, e.g. to proteins which are in vivo binding partners of that DNA-PKcs-like polypeptide.
Accordingly, in still further aspects, the present invention provides for assays and methods of screening for a substance which modulates, e.g. inhibits, reduces or interferes with, binding of a DNA-PKcs-like polypeptide to an EGGDVDDLLDMI polypeptide, and for the use of DNA-PKcs-like polypeptides and/or EGGDVDDLLDMI polypeptides in these screening methods and assays.
Such a method may include:
(i) bringing a DNA-PKcs-like polypeptide into contact with an EGGDVDDLLDMI polypeptide in the presence of one or more test substances;
(ii) determining binding of the DNA-PKcs-like polypeptide to the EGGDVDDLLDMI polypeptide.
Binding in the presence of a test substance may be compared with binding of the DNA-PKcs-like polypeptide to the EGGDVDDLLDMI polypeptide in comparable reaction medium and conditions in the absence of a test substance. A test substance able to modulate the binding may be identified.
Agents which modulate e.g. increase or potentiate interaction between a DNA-PKcs-like polypeptide and an EGGDVDDLLDMI polypeptide may be identified using conditions which, in the absence of a positively-testing agent, prevent or impair the substances interacting.
A difference in the binding of the DNA-PKcs-like polypeptide to the EGGDVDDLLDMI polypeptide between the treated and untreated conditions is indicative of a modulating effect of the relevant test substance(s).
Further aspects of the invention provide assay methods for identifying or obtaining agents which modulate, especially interfere with, interaction between Ku70 or Ku80 and a binding partner, via a Homology Region as identified herein.
Such methods may comprise
(i) bringing a Ku Homology Region Peptide into contact with a binding partner in the presence of one or more test substances;
(ii) determining binding of the Ku Homology Region Peptide to the binding partner.
Binding may be determined by any suitable method known to those skilled in the art. Examples of such methods are described below.
In any assay method according to the invention, the amount of test substance or compound which may be added to an assay of the invention will normally be determined by trial and error depending upon the type of compound used. Typically, from about 0.001 nM to 1 mM or more concentrations of putative inhibitor compound may be used, for example from 0.01 nM to 100 xcexcM, e.g. 0.1 to 50 xcexcM, such as about 10 xcexcM. Greater concentrations may be used when a peptide is the test substance. Even a molecule which has a weak effect may be a useful lead compound for further investigation and development.
Compounds which may be screened may be natural or synthetic chemical compounds used in drug screening programmes. Extracts of plants, microbes or other organisms, which contain several characterised or uncharacterised components may also be used.
It is worth noting that combinatorial library technology provides an efficient way of testing a potentially vast number of different substances for ability to modulate an interaction. Such libraries and their use are known in the art, for all manner of natural products, small molecules and peptides, among others. The use of peptide libraries may be preferred in certain circumstances.
In various aspects the present invention provides a modulator identified by a screening method of the invention, e.g. a substance which interferes with or interrupts, increases or potentiates binding of a DNA-PKcs-like polypeptide (e.g. DNA-PKcs) to a target EGGDVDDLLDMI polypeptide (e.g. Ku80 or an EGGDVDDLLDMI polypeptide of the present invention), or Ku70 and/or Ku80 to each other or a binding partner (via a Homology Region).
Following identification of a modulator, the substance may be purified and/or investigated further and/or manufactured. A modulator may be used to obtain peptidyl or non-peptidyl mimetics, e.g. by methods well known to those skilled in the art and discussed herein. It may be used in a therapeutic context as discussed below.
One class of modulators comprises peptides comprising the amino acid sequence EGGDVDDLLDMI (SEQ ID NO:1) or variants thereof, which peptides have the ability to bind to a DNA-PKCS-like polypeptide. A further class of modulators comprises peptide fragments of DNA-PKCS-like polypeptides, or variants thereof, particularly fragments of DNA-PKCS-like polypeptides which contain sequence motifs that have been identified (e.g. in a screening method of the present invention) as being involved in binding of the DNA-PKCS-like polypeptide to an EGGDVDDLLDMI sequence (SEQ ID NO:1).
Suitable peptide modulators are those which bind to Ku80 or to an EGGDVDDLLDMI polypeptide shown in FIG. 4 and/or which have a length of 50-55, 55-60, 60-65, 65-70, 70-75, 75-80, 80-85, 85-90, 90-95, 95-100, or more than 100 amino acids. Nucleic acid encoding such peptide frgaments, vectors and host cells containing such nucleic acid, and methods of expressing nucleic acid encoding such fragments are further aspects of the present invention. The discussion included herein relating to the construction, use and expression of peptides and polypeptides and encoding nucleic acid applies to such embodiments as appropriate.
Antibodies directed to the a peptide disclosed herein, a Ku Homology Region peptide or EGGDVDDLLDMI, or to a sequence in a DNA-PKcs-like polypeptide identified (e.g. in a method of the present invention) as being involved in an interaction with an EGGDVDDLLDMI polypeptide, form further classes of putative inhibitor compounds and represent individual aspects of the invention in their own right. Candidate inhibitor antibodies may be characterised and their binding regions determined to provide single chain antibodies and fragments thereof which are responsible for disrupting the binding.
Antibodies in accordance with and/or for use in the present invention may be obtained using techniques which are standard in the art. Methods of producing antibodies include immunising a mammal (e.g. mouse, rat, rabbit, horse, goat, sheep or monkey) with a polypeptide or fragment thereof, e.g. with a EGGDVDDLLDMI polypeptide (e.g. with the polypeptide EGGDVDDLLDMI (SEQ ID NO:1) or a variant thereof), or with a DNA-PKCS-like polypeptide, or with a Ku Homology Region Peptide. Antibodies may be obtained from immunised animals using any of a variety of techniques known in the art, and screened, preferably using binding of antibody to antigen of interest. For instance, Western blotting techniques or immunoprecipitation may be used (Armitage et al., 1992, Nature 357: 80-82). Isolation of antibodies and/or antibody-producing cells from an animal may be accompanied by a step of sacrificing the animal.
As an alternative or supplement to immunising a mammal with a peptide, an antibody specific for a peptide or polypeptide of interest may be obtained from a recombinantly produced library of expressed immunoglobulin variable domains, e.g. using lambda bacteriophage or filamentous bacteriophage which display functional immunoglobulin binding domains on their surfaces; for instance see WO92/01047. The library may be naive, that is constructed from sequences obtained from an organism which has not been immunised with the relevant peptide or polypeptide (or fragments thereof), or may be one constructed using sequences obtained from an organism which has been exposed to the antigen of interest.
Antibodies according to and/or for use in the present invention may be modified in a number of ways. Indeed the term xe2x80x9cantibodyxe2x80x9d should be construed as covering any binding substance having a binding domain with the required specificity. Thus the invention covers antibody fragments, derivatives, functional equivalents and homologues of antibodies, including synthetic molecules and molecules whose shape mimicks that of an antibody enabling it to bind an antigen or epitope.
Example antibody fragments, capable of binding an antigen or other binding partner are the Fab fragment consisting of the VL, VH, Cl and CH1 domains; the Fd fragment consisting of the VH and CH1 domains; the Fv fragment consisting of the VL and VH domains of a single arm of an antibody; the dAb fragment which consists of a VH domain; isolated CDR regions and F(abxe2x80x2)2 fragments, a bivalent fragment including two Fab fragments linked by a disulphide bridge at the hinge region. Single chain Fv fragments are also included.
A hybridoma producing a monoclonal antibody according to the present invention may be subject to genetic mutation or other changes. It will further be understood by those skilled in the art that a monoclonal antibody can be subjected to the techniques of recombinant DNA technology to produce other antibodies or chimeric molecules which retain the specificity of the original antibody. Such techniques may involve introducing DNA encoding the immunoglobulin variable region, or the complementarity determining regions (CDRs), of an antibody to the constant regions, or constant regions plus framework regions, of a different immunoglobulin. See, for instance, EP184187A, GB 2188638A or EP-A-0239400. Cloning and expression of chimeric antibodies are described in EP-A-0120694 and EP-A-0125023.
Hybridomas capable of producing antibody with desired binding characteristics are within the scope of the present invention, as are host cells, eukaryotic or prokaryotic, containing nucleic acid encoding antibodies (including antibody fragments) and capable of their expression. The invention also provides methods of production of the antibodies including growing a cell capable of producing the antibody under conditions in which the antibody is produced, and preferably secreted.
The reactivities of antibodies on a sample may be determined by any appropriate means. Tagging with individual reporter molecules is one possibility. The reporter molecules may directly or indirectly generate detectable, and preferably measurable, signals. The linkage of reporter molecules may be directly or indirectly, covalently, e.g. via a peptide bond or non-covalently. Linkage via a peptide bond may be as a result of recombinant expression of a gene fusion encoding antibody and reporter molecule. The mode of determining binding is not a feature of the present invention and those skilled in the art are able to choose a suitable mode according to their preference and general knowledge.
Antibodies may also be used in purifying and/or isolating a polypeptide, e.g. an EGGDVDDLLDMI polypeptide or a Ku polypeptide or DNA-PKCS-like polypeptide for instance following production of the polypeptide by expression from encoding nucleic acid therefor. The antibodies may be respectively directed to the amino acid sequence EGGDVDDLLDMI (SEQ ID NO:1), to a Ku Homology Region Peptide, or to a region of a DNA-PKCS-like polypeptide identified (e.g. by a method of the present invention) as having the ability to interact with an EGGDVDDLLDMI polypeptide. Antibodies may be useful in a therapeutic context (which may include prophylaxis) e.g. to disrupt binding of a DNA-PKCS-like polypeptide to an EGGDVDDLLDMI polypeptide with a view to inhibiting the activity of either component. Antibodies can for instance be micro-injected into cells, e.g. at a tumour site, subject to radio- and/or chemo-therapy (as discussed already above). Antibodies may be employed in accordance with the present invention for other therapeutic and non-therapeutic purposes which are discussed elsewhere herein.
In a further aspect, the present invention provides the use of a peptide as disclosed herein in a method of designing a peptide or non-peptidyl mimetic of the polypeptide. A mimetic may be able to bind to a DNA-PKCS-like polypeptide and/or modulate interaction between a DNA-PKCS-like polypeptide and an EGGDVDDLLDMI polypeptide. A EGGDVDDLLDMI polypeptide used in such a method may be the amino acid sequence EGGDVDDLLDMI (SEQ ID NO:1) or a variant thereof, or an EGGDVDDLLDMI polypeptide of the present invention, e.g. one shown in FIG. 4.
The present invention similarly provides for the use of a DNA-PKCS-like polypeptide, for example DNA-PKCS, particularly a DNA-PKCS-like polypeptide identified as having the ability to bind to the amino acid sequence EGGDVDDLLDMI (SEQ ID NO:1), in a method of designing a peptide or non-peptidyl mimetic of a DNA-PKCS-like polypeptide, which mimetic is able to bind to a given EGGDVDDLLDMI polypeptide, e.g. to Ku80.
Accordingly, the present invention provides a method of designing a mimetic of a EGGDVDDLLDMI polypeptide which has the biological activity of binding to a DNA-PKcs-like polypeptide (e.g. DNA-PKcs), or a method of designing a mimetic of a DNA-PKcs-like polypeptide which has biological activity of binding to a target EGGDVDDLLDMI polypeptide (e.g. to Ku80 and/or to to an EGGDVDDLLDMI polypeptide of the present invention), or a method of designing a mimetic of a Ku Homology Region Peptide, said method comprising:
(i) analysing a substance having the biological activity to determine the amino acid residues essential and important for the activity to define a pharmacophore; and,
(ii) modelling the pharmacophore to design and/or screen candidate mimetics having the biological activity.
Suitable modelling techniques are known in the art. This includes the study of the bonding between peptides or polypeptides and to design compounds which contain functional groups arranged in such a manner that they could reproduced that bonding.
The designing of mimetics to a known pharmaceutically active compound is a known approach to the development of pharmaceuticals based on a xe2x80x9cleadxe2x80x9d compound. This might be desirable where the active compound is difficult or expensive to synthesise or where it is unsuitable for a particular method of administration, for instance polypeptides of the invention may not be well suited as active agents for oral compositions as they tend to be quickly degraded by proteases in the alimentary canal.
There are several steps commonly taken in the design of a mimetic from a compound having a given target property. Firstly, the particular parts of the compound that are critical and/or important in determining the target property are determined. In the case of a peptide, this can be done by systematically varying the amino acid residues in the peptide, e.g. by substituting each residue in turn. These parts or residues constituting the active region of the compound are known as its xe2x80x9cpharmacophorexe2x80x9d.
Once the pharmacophore has been found, its structure is modelled according to its physical properties, e.g. stereochemistry, bonding, size and/or charge, using data from a range of sources, e.g. spectroscopic techniques, X-ray diffraction data and NMR. Computational analysis, similarity mapping (which models the charge and/or volume of a pharmacophore, rather than the bonding between atoms) and other techniques can be used in this modelling process.
In a variant of the above approach, the three-dimensional structure of a ligand and its binding partner are modelled. This can be especially useful where the ligand and/or binding partner change conformation on binding, allowing the model to take account of this the design of the mimetic.
A template molecule is then selected onto which chemical groups which mimic the pharmacophore can be grafted. The template molecule and the chemical groups grafted on to it can conveniently be selected so that the mimetic is easy to synthesise, is likely to be pharmacologically acceptable, and does not degrade in vivo, while retaining the biological activity of the lead compound. The mimetic or mimetics found by this approach can then be screened to see whether they have the target property, or to what extent they exhibit it. Further optimisation or modification can then be carried out to arrive at one or more final mimetics for in vivo or clinical testing.
The mimetic or mimetics found by any of the approaches described herein may be used in the assay methods of the present invention to determine whether they have the ability to bind to the relevant compound, e.g. to a DNA-PKcs-like polypeptide (e.g. DNA-PKCS) or to a target EGGDVDDLLDMI polypeptide(e.g. to Ku80 and/or to an EGGDVDDLLDMI polypeptide of the present invention).
Mimetics obtained by a method of the invention form a further aspect of the invention.
The invention further provides various therapeutic methods and uses of one or more substances selected from (i) an EGGDVDDLLDMI polypeptide, (e.g. an EGGDVDDLLDMI polypeptide according to the present invention, a fragment of Ku80 comprising the sequence EGGDVDDLLDMI (SEQ ID NO:1), an EGGDVDDLLDMI polypeptide identified in accordance with a method of the present invention); (ii) a DNA-PKCS-like polypeptide, (e.g. a variant of DNA-PKCS, e.g. fragment of DNA-PKCS, a DNA-PKCS-like polypeptide identified by a screening method of the present invention); (iii) any peptide or polypeptide disclosed herein, e.g. a Ku Homology Region Peptide; (iv) a modulator identified by a screening method of the present invention; (v) a mimetic of any of the above substances which can bind to a DNA-PKCS-like polypeptide or EGGDVDDLLDMI polypeptide.
The therapeutic/prophylactic purpose of such a method or use may be the modulation, e.g. disruption or interference, of the binding of a DNA-PKcs-like polypeptide to an EGGDVDDLLDMI polypeptide, or a Ku Homology Region to a binding partner, e.g. to modulate any activity mediated by virtue, of such binding.
The therapeutic/prophylactic purpose may for example be:
(i) Chemotherapy and/or radiotherapy, e.g. sensitising tumours or other cells to radiation or chemotherapy, e.g. modulating (for example inhibiting) interactions of Ku70, Ku80 and/or DNA-PKcs leading to e.g. impairment of tumour proliferation and/or growth;
(ii) Modulation (e.g. inhibition) of infection of pathogens (e.g. viruses) whose infectivity is influenced by Ku and/or DNA-PKCS, e.g. inhibition of retroviral integration;
(iii) Controlling cell e.g. tumour growth via modulation of telomere function;
(iv) Modulating V(D)J recombination or other genomic rearrangement that employs Ku and/or DNA-PKcs.
In various further aspects the present invention thus provides a pharmaceutical composition, medicament, drug or other composition for such a purpose, the composition comprising one or more of the substances set out above, the use of such a substance in a method of medical treatment, a method comprising administration of such a substance or composition to a patient, e.g. for treatment (which may include preventative treatment) of a medical condition, e.g. a condition associated with a defect or disorder in DNA repair, or cell cycle control, e.g. for treatment of a disorder of cellular proliferation such as cancer, use of such a substance in the manufacture of a composition, medicament or drug for administration for such a purpose, e.g. for treatment of a proliferative disorder, and a method of making a pharmaceutical composition comprising admixing such a substance with a pharmaceutically acceptable excipient, vehicle or carrier, and optionally other ingredients.
The substances may be used as sole active agents or in combination with one another or with any other active substance, e.g. for anti-tumour therapy another anti-tumour compound or therapy, such as radiotherapy or chemotherapy. Whatever the substance used in a method of medical treatment of the present invention, administration is preferably in a xe2x80x9cprophylactically effective amountxe2x80x9d or a xe2x80x9ctherapeutically effective amountxe2x80x9d (as the case may be, although prophylaxis may be considered therapy), this being sufficient to show benefit to the individual. The actual amount administered, and rate and time-course of administration, will depend on the nature and severity of what is being treated. Prescription of treatment, e.g. decisions on dosage etc, is within the responsibility of general practioners and other medical doctors.
A substance or composition may be administered alone or in combination with other treatments, either simultaneously or sequentially dependent upon the condition to be treated, e.g. cancer.
Pharmaceutical compositions according to the present invention, and for use in accordance with the present invention, may include, in addition to active ingredient, a pharmaceutically acceptable excipient, carrier, buffer, stabiliser or other materials well known to those skilled in the art. Such materials should be non-toxic and should not interfere with the efficacy of the active ingredient. The precise nature of the carrier or other material will depend on the route of administration, which may be oral, or by injection, e.g. cutaneous, subcutaneous or intravenous.
Pharmaceutical compositions for oral administration may be in tablet, capsule, powder or liquid form. A tablet may include a solid carrier such as gelatin or an adjuvant. Liquid pharmaceutical compositions generally include a liquid carrier such as water, petroleum, animal or vegetable oils, mineral oil or synthetic oil. Physiological saline solution, dextrose or other saccharide solution or glycols such as ethylene glycol, propylene glycol or polyethylene glycol may be included.
For intravenous, cutaneous or subcutaneous injection, or injection at the site of affliction, the active ingredient will be in the form of a parenterally acceptable aqueous solution which is pyrogen-free and has suitable pH, isotonicity and stability. Those of relevant skill in the art are well able to prepare suitable solutions using, for example, isotonic vehicles such as Sodium Chloride Injection, Ringer""s Injection, Lactated Ringer""s Injection. Preservatives, stabilisers, buffers, antioxidants and/or other additives may be included, as required.
Liposomes, particularly cationic liposomes, may be used in carrier formulations.
Examples of techniques and protocols mentioned above can be found in Remington""s Pharmaceutical Sciences, 16th edition, Osol, A. (ed), 1980.
The substance or composition may be administered in a localised manner to a tumour site or other desired site or may be delivered in a manner in which it targets tumour or other cells.
Targeting therapies may be used to deliver the active substance more specifically to certain types of cell, by the use of targeting systems such as antibody or cell specific ligands. Targeting may be desirable for a variety of reasons, for example if the agent is unacceptably toxic, or if it would otherwise require too high a dosage, or if it would not otherwise be able to enter the target cells.
Instead of administering such substances directly, they may be produced in the target cells by expression from an encoding nucleic acid introduced into the cells, e.g. from a viral vector. The vector may be targeted to the specific cells to be treated, or it may contain regulatory elements which are switched on more or less selectively by the target cells.
Nucleic acid encoding the substance e.g. a polypeptide able to modulate, e.g. interfere with, the binding of a DNA-PKcs-like polypeptide to an EGGDVDDLLDMI polypeptide may thus be used in methods of gene therapy, for instance in treatment of individuals, e.g. with the aim of preventing or curing (wholly or partially) a disorder.
Vectors such as viral vectors have been used in the prior art to introduce nucleic acid into a wide variety of different target cells. Typically the vectors are exposed to the target cells so that transfection can take place in a sufficient proportion of the cells to provide a useful therapeutic or prophylactic effect from the expression of the desired polypeptide. The transfected nucleic acid may be permanently incorporated into the genome of each of the targeted cells, providing long lasting effect, or alternatively the treatment may have to be repeated periodically.
A variety of vectors, both viral vectors and plasmid vectors, are known in the art, see U.S. Pat. No. 5,252,479 and WO 93/07282. In particular, a number of viruses have been used as gene transfer vectors, including papovaviruses, such as SV40, vaccinia virus, herpesviruses, including HSV and EBV, and retroviruses. Many gene therapy protocols in the prior art have used disabled murine retroviruses.
As an alternative to the use of viral vectors in gene therapy other known methods of introducing nucleic acid into cells includes mechanical techniques such as microinjection, transfer mediated by liposomes and receptor-mediated DNA transfer.
Receptor-mediated gene transfer, in which the nucleic acid is linked to a protein ligand via polylysine, with the ligand being specific for a receptor present on the surface of the target cells, is an example of a technique for specifically targeting nucleic acid to particular cells.
A peptide, polypeptide or other substance according to the present invention, e.g. a nucleic acid molecule which encodes a peptide or polypeptide, may be provided in a kit, e.g. sealed in a suitable container which protects its contents from the external environment. Such a kit may include instructions for use.
In still further aspects the present invention provides for the purification of a DNA-PKcs-like polypeptide or protein containing a DNA-PKcs-like polypeptide, or for the purification of an EGGDVDDLLDMI polypeptide. The invention also provides for a purified DNA-PKcs-like polypeptide or protein containing a DNA-PKcs-like polypeptide and a purified EGGDVDDLLDMI polypeptide. The purified protein or polypeptide may be about,10% pure, more preferably about 20% pure, more preferably about 30% pure, more preferably about 40% pure, more preferably about 50% pure, more preferably about 60% pure, more preferably about 70% pure, more preferably about 80% pure, more preferably about 90% pure, more preferably about 95% pure, or substantially pure.
The present invention thus provides a method of purifying a DNA-PKCS-like polypeptide or protein containing a DNA-PKCS-like polypeptide, the method including contacting the DNA-PKCS-like polypeptide with an EGGDVDDLLDMI peptide. The present inventors have already shown that the DNA-PKCS-like polypeptide human DNA-PKCS can be purified to virtual homogeneity in a single step using a peptide of amino acid sequence EGGDVDDLLDMI (SEQ ID NO:1).
A mixture of material including a DNA-PKcs-like polypeptide or protein containing a DNA-PKcs-like polypeptide may be contacted against immobilised EGGDVDDLLDMI polypeptide (e.g. immobilised either covalently or non-covalently such as via a specific binding molecule such as streptavidin or biotin) and molecules which do not bind to the phosphopeptide are washed off.
Likewise, the invention provides a method of purifying an EGGDVDDLLDMI polypeptide, the method including contacting material containing the polypeptide with a DNA-PKcs-like polypeptide.
Preferred EGGDVDDLLDMI polypeptides and DNA-PKcs-like polypeptides for use in methods of the invention are discussed elsewhere herein.
The DNA-PKcs-like polypeptide or protein containing a DNA-PKcs-like polypeptide which is contacted with the EGGDVDDLLDMI polypeptide, or the EGGDVDDLLDMI polypeptide which is contacted with the DNA-PKcs-like polypeptide, in a purification method of the present invention, may be in a mixture of molecules, such as a cellular extract, such as a normal cell of an organism such as a human or a recombinant host cell expressing the protein or polypeptide from encoding DNA, such as a bacterial, eukaryotic (e.g. mammalian or yeast) or insect cell, such as in a baculovirus expression system. Purification may follow production of such a polypeptide recombinantly in a suitable expression system, such as a cell, by expression from encoding nucleic acid. Following purification, the DNA-PKcs-like polypeptide or protein containing the DNA-PKcs-like polypeptide or the EGGDVDDLLDMI polypeptide may be used as desired, e.g. in an assay for an agent which modulates its activity, e.g. binding, in raising or obtaining a specific antibody or other binding molecule, or in a therapeutic context.
Methods of determining the binding of a DNA-PKCS-like polypeptide to an EGGDVDDLLDMI polypeptide, of identifying a. DNA-PKcs-like polypeptide, of screening for an EGGDVDDLLDMI polypeptide able to bind to a DNA-PKcs-like polypeptide, and of screening for an agent able to modulate binding of a DNA-PKcs-like polypeptide to an EGGDVDDLLDMI polypeptide, include methods in which a suitable end-point is used to assess binding.
Binding may be determined by any number of techniques known in the art, qualitative or quantitative. They include techniques such as radioimmunosassay, co-immunoprecipitation, scintillation proximetry assay and ELISA methods.
Binding of a DNA-PKCS-like polypeptide (e.g. DNA-PKCS) to a target EGGDVDDLLDMI polypeptide (e.g. to a peptide of amino acid sequence EGGDVDDLLDMI (SEQ ID NO:1)) may be studied by labelling either one with a detectable label and bringing it into contact with the other which may have been immobilised on a solid support.
Suitable detectable labels, especially for peptidyl substances include 35S-methionine which may be incorporated into recombinantly produced peptides and polypeptides. Recombinantly produced peptides and polypeptides may also be expressed as a fusion protein containing an epitope which can be labelled with an antibody.
The polypeptide which is immobilized on a solid support may be immobilized using an antibody against that polypeptide bound to a solid support or via other technologies which are known per se. A preferred in vitro interaction may utilise a fusion polypeptide including glutathione-S-transferase (GST). This may be immobilized on glutathione agarose beads. In an in vitro assay format of the type described above a test modulator can be assayed by determining its ability to diminish the amount of labelled polypeptide (e.g. labelled EGGDVDDLLDMI polypeptide) which binds to the immobilized GST-fusion polypeptide (e.g. immobilised fusion polypeptide of GST and a DNA-PKcs-like polypeptide). This may be determined by fractionating the glutathione-agarose beads by SDS-polyacrylamide gel electrophoresis. Alternatively, the beads may be rinsed to remove unbound polypeptide and the amount of polypeptide which has bound can be determined by counting the amount of label present in, for example, a suitable scintillation counter.
Binding or interaction of a DNA-PKcs-like polypeptide and an EGGDVDDLLDMI polypeptide may also be determined using a two-hybrid assay.
For example, a DNA-PKcs-like polypeptide polypeptide or an EGGDVDDLLDMI polypeptide may be fused to a DNA binding domain such as that of the yeast transcription factor GAL4. The GAL4 transcription factor includes two functional domains. These domains are the DNA binding domain (GAL4DBD) and the GAL4 transcriptional activation domain (GAL4TAD). By fusing the DNA-PKcs-like polypeptide to one of those domains, and an EGGDVDDLLDMI polypeptide to the respective counterpart, a functional GAL4 transcription factor is restored only when the DNA-PKcs-like polypeptide and EGGDVDDLLDMI polypeptide interact. Thus, interaction of these polypeptides may be measured by the use of a reporter gene linked to a GAL4 DNA binding site which is capable of activating transcription of said reporter gene.
This two hybrid assay format is described by Fields and Song, 1989, Nature 340; 245-246. It can be used in both mammalian cells and in yeast. Other combinations of DNA binding domain and transcriptional activation domain are available in the art and may be preferred, such as the LexA DNA binding domain and the VP60 transcriptional activation domain.
When looking for substances which interfere with binding of (for example) a DNA-PKcs-like polypeptide to an EGGDVDDLLDMI polypeptide, a DNA-PKcs-like polypeptide or a EGGDVDDLLDMI polypeptide may be employed as a fusion with (e.g.) the LexA DNA binding domain, and the counterpart polypeptide containing the DNA-PKxcs-like polypeptide or EGGDVDDLLDMI polypeptide as a fusion with (e.g.) VP60. An expression cassette may be used to express a test peptide within a host cell. The expression cassette may be one vector from a library of expression vectors which encode a diverse range of peptides. A reduction in reporter gene expression (e.g. in the case of xcex2-galactosidase a weakening of the blue colour) results from the expression of a peptide which disrupts the DNA-PKcs-like polypeptide/EGGDVDDLLDMI polypeptide interaction, which interaction is required for transcriptional activation of the xcex2-galactosidase gene. Where a test substance is not peptidyl and may not be expressed from encoding nucleic acid within a said third expression cassette, a similar system may be employed with the test substance supplied exogenously.
An assay or screening method according to the present invention may thus take the form of an in vivo assay. The end-point of an in vivo assay, that is to say the property which is determined in order to assess the binding of a DNA-PKcs-like polypeptide to an EGGDVDDLLDMI polypeptide (e.g. to assess whether a test agent has an effect on the binding of a DNA-PKcs-like polypeptide to a EGGDVDDLLDMI polypeptide) may be a biological activity of the DNA-PKcs-like polypeptide or a biological activity of the EGGDVDDLLDMI polypeptide, whichever is appropriate. As noted, those skilled in the art well appreciate the need for and design of appropriate controls for validation of results.
As end points for in vivo assays employing human DNA-PKcs or a homologue/orthologue thereof, and/or Ku, the effect on DNA repair, cell viability, cell killing, radiosensitivity, V(D)J recomination, cell cycle arrest. In yeast, P element transposition, and mating type switching may be measured. Suitable methods are known to those skilled in the art.